Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACβ) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.

Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the trans-Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles.IMPORTANCE Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes.

Interferon-inducible guanylate-binding proteins (GBPs) promote cell-intrinsic defense through host cell death. GBPs target pathogens and pathogen-containing vacuoles and promote membrane disruption for release of microbial molecules that activate inflammasomes. GBP1 mediates pyroptosis or atypical apoptosis of Salmonella Typhimurium (STm)- or Toxoplasma gondii (Tg)- infected human macrophages, respectively. The pathogen-proximal detection-mechanisms of GBP1 remain poorly understood, as humans lack functional immunity-related GTPases (IRGs) that assist murine Gbps. Here, we establish that GBP1 promotes the lysis of Tg-containing vacuoles and parasite plasma membranes, releasing Tg-DNA. In contrast, we show GBP1 targets cytosolic STm and recruits caspase-4 to the bacterial surface for its activation by lipopolysaccharide (LPS), but does not contribute to bacterial vacuole escape. Caspase-1 cleaves and inactivates GBP1, and a cleavage-deficient GBP1D192E mutant increases caspase-4-driven pyroptosis due to the absence of feedback inhibition. Our studies elucidate microbe-specific roles of GBP1 in infection detection and its triggering of the assembly of divergent caspase signaling platforms.

Intestine function relies on the strong polarity of intestinal epithelial cells and the array of microvilli forming a brush border at their luminal pole. Combining a genetic RNA interference (RNAi) screen with in vivo super-resolution imaging in the Caenorhabditiselegans intestine, we found that the V0 sector of the vacuolar ATPase (V0-ATPase) controls a late apical trafficking step, involving Ras-related protein 11 (RAB-11)+ endosomes and the N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) synaptosome-associated protein 29 (SNAP-29), and is necessary to maintain the polarized localization of both apical polarity modules and brush border proteins. We show that the V0-ATPase pathway also genetically interacts with glycosphingolipids and clathrin in enterocyte polarity maintenance. Finally, we demonstrate that silencing of the V0-ATPase fully recapitulates the severe structural, polarity and trafficking defects observed in enterocytes from individuals with microvillus inclusion disease (MVID) and use this new in vivo MVID model to follow the dynamics of microvillus inclusions. Thus, we describe a new function for V0-ATPase in apical trafficking and epithelial polarity maintenance and the promising use of the C. elegans intestine as an in vivo model to better understand the molecular mechanisms of rare genetic enteropathies.

Super-resolution light microscopy, correlative light and electron microscopy, and volume electron microscopy are revolutionising the way in which biological samples are examined and understood. Here, we combine these approaches to deliver super-accurate correlation of fluorescent proteins to cellular structures. We show that YFP and GFP have enhanced blinking properties when embedded in acrylic resin and imaged under partial vacuum, enabling in vacuo single molecule localisation microscopy. In conventional section-based correlative microscopy experiments, the specimen must be moved between imaging systems and/or further manipulated for optimal viewing. These steps can introduce undesirable alterations in the specimen, and complicate correlation between imaging modalities. We avoided these issues by using a scanning electron microscope with integrated optical microscope to acquire both localisation and electron microscopy images, which could then be precisely correlated. Collecting data from ultrathin sections also improved the axial resolution and signal-to-noise ratio of the raw localisation microscopy data. Expanding data collection across an array of sections will allow 3-dimensional correlation over unprecedented volumes. The performance of this technique is demonstrated on vaccinia virus (with YFP) and diacylglycerol in cellular membranes (with GFP).

The eukaryotic genome is organized in a three-dimensional structure called chromatin, constituted by DNA and associated proteins, the majority of which are histones. Post-translational modifications of histone proteins greatly influence chromatin structure and regulate many DNA-based biological processes. Methylation of lysine 36 of histone 3 (H3K36) is a post-translational modification functionally relevant during early steps of DNA damage repair. Here, we show that the JMJD-5 regulates H3K36 di-methylation and it is required at late stages of double strand break repair mediated by homologous recombination. Loss of jmjd-5 results in hypersensitivity to ionizing radiation and in meiotic defects, and it is associated with aberrant retention of RAD-51 at sites of double strand breaks. Analyses of jmjd-5 genetic interactions with genes required for resolving recombination intermediates (rtel-1) or promoting the resolution of RAD-51 double stranded DNA filaments (rfs-1 and helq-1) suggest that jmjd-5 prevents the formation of stalled postsynaptic recombination intermediates and favors RAD-51 removal. As these phenotypes are all recapitulated by a catalytically inactive jmjd-5 mutant, we propose a novel role for H3K36me2 regulation during late steps of homologous recombination critical to preserve genome integrity.

The intestinal brush border is made of an array of microvilli that increases the membrane surface area for nutrient processing, absorption and host defense. Studies on mammalian cultured epithelial cells have uncovered some of the molecular players and physical constraints required to establish this apical specialized membrane. However, the building and maintenance of a brush border in vivo has not yet been investigated in detail. Here, we combined super-resolution imaging, transmission electron microscopy and genome editing in the developing nematode Caenorhabditis elegans to build a high-resolution and dynamic localization map of known and new brush border markers. Notably, we show that microvilli components are dynamically enriched at the apical membrane during microvilli outgrowth and maturation, but become highly stable once microvilli are built. This new toolbox will be instrumental for understanding the molecular processes of microvilli growth and maintenance in vivo, as well as the effect of genetic perturbations, notably in the context of disorders affecting brush border integrity.

Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging.

This paper describes an unsupervised algorithm, which segments the nuclear envelope of HeLa cells imaged by Serial Block Face Scanning Electron Microscopy. The algorithm exploits the variations of pixel intensity in different cellular regions by calculating edges, which are then used to generate superpixels. The superpixels are morphologically processed and those that correspond to the nuclear region are selected through the analysis of size, position, and correspondence with regions detected in neighbouring slices. The nuclear envelope is segmented from the nuclear region. The three-dimensional segmented nuclear envelope is then modelled against a spheroid to create a two-dimensional (2D) surface. The 2D surface summarises the complex 3D shape of the nuclear envelope and allows the extraction of metrics that may be relevant to characterise the nature of cells. The algorithm was developed and validated on a single cell and tested in six separate cells, each with 300 slices of 2000 × 2000 pixels. Ground truth was available for two of these cells, i.e., 600 hand-segmented slices. The accuracy of the algorithm was evaluated with two similarity metrics: Jaccard Similarity Index and Mean Hausdorff distance. Jaccard values of the first/second segmentation were 93%/90% for the whole cell, and 98%/94% between slices 75 and 225, as the central slices of the nucleus are more regular than those on the extremes. Mean Hausdorff distances were 9/17 pixels for the whole cells and 4/13 pixels for central slices. One slice was processed in approximately 8 s and a whole cell in 40 min. The algorithm outperformed active contours in both accuracy and time.

Epithelial tubes are essential components of metazoan organ systems that control the flow of fluids and the exchange of materials between body compartments and the outside environment. The size and shape of the central lumen confer important characteristics to tubular organs and need to be carefully controlled. Here, we identify the small coiled-coil protein BBLN-1 as a regulator of lumen morphology in the C. elegans intestine. Loss of BBLN-1 causes the formation of bubble-shaped invaginations of the apical membrane into the cytoplasm of intestinal cells and abnormal aggregation of the subapical intermediate filament (IF) network. BBLN-1 interacts with IF proteins and localizes to the IF network in an IF-dependent manner. The appearance of invaginations is a result of the abnormal IF aggregation, indicating a direct role for the IF network in maintaining lumen homeostasis. Finally, we identify bublin (BBLN) as the mammalian ortholog of BBLN-1. When expressed in the C. elegans intestine, BBLN recapitulates the localization pattern of BBLN-1 and can compensate for the loss of BBLN-1 in early larvae. In mouse intestinal organoids, BBLN localizes subapically, together with the IF protein keratin 8. Our results therefore may have implications for understanding the role of IFs in regulating epithelial tube morphology in mammals.

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure.

Crumbs proteins are evolutionarily conserved transmembrane proteins with essential roles in promoting the formation of the apical domain in epithelial cells. The short intracellular tail of Crumbs proteins are known to interact with several proteins, including the scaffolding protein PALS1 (protein associated with LIN7, Stardust in Drosophila). PALS1 in turn binds to a second scaffolding protein PATJ (PALS1-associated tight junction protein) to form the core Crumbs/PALS1/PATJ complex. While essential roles in epithelial organization have been shown for Crumbs proteins in Drosophila and mammalian systems, the three Caenorhabditis elegans crumbs genes are dispensable for epithelial polarization and development. Here, we investigated the presence and function of PALS1 and PATJ orthologs in C. elegans. We identified MAGU-2 as the C. elegans ortholog of PALS1 and show that MAGU-2 interacts with all three Crumbs proteins and localizes to the apical membrane domain of intestinal epithelial cells in a Crumbs-dependent fashion. Similar to crumbs mutants, magu-2 deletion showed no epithelial polarity defects. We also identified MPZ-1 as a candidate ortholog of PATJ based on the physical interaction with MAGU-2 and sequence similarity with PATJ proteins. However, MPZ-1 is not broadly expressed in epithelial tissues and, therefore, not likely a core component of the C. elegans Crumbs complex. Finally, we show overexpression of the Crumbs proteins EAT-20 or CRB-3 can lead to apical membrane expansion in the intestine. Our results shed light on the composition of the C. elegans Crumbs complex and indicate that the role of Crumbs proteins in promoting apical domain formation is conserved.

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of "pseudoschizonts," which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

The quantitative study of cell morphology is of great importance as the structure and condition of cells and their structures can be related to conditions of health or disease. The first step towards that, is the accurate segmentation of cell structures. In this work, we compare five approaches, one traditional and four deep-learning, for the semantic segmentation of the nuclear envelope of cervical cancer cells commonly known as HeLa cells. Images of a HeLa cancer cell were semantically segmented with one traditional image-processing algorithm and four three deep learning architectures: VGG16, ResNet18, Inception-ResNet-v2, and U-Net. Three hundred slices, each 2000 × 2000 pixels, of a HeLa Cell were acquired with Serial Block Face Scanning Electron Microscopy. The first three deep learning architectures were pre-trained with ImageNet and then fine-tuned with transfer learning. The U-Net architecture was trained from scratch with 36, 000 training images and labels of size 128 × 128. The image-processing algorithm followed a pipeline of several traditional steps like edge detection, dilation and morphological operators. The algorithms were compared by measuring pixel-based segmentation accuracy and Jaccard index against a labelled ground truth. The results indicated a superior performance of the traditional algorithm (Accuracy = 99%, Jaccard = 93%) over the deep learning architectures: VGG16 (93%, 90%), ResNet18 (94%, 88%), Inception-ResNet-v2 (94%, 89%), and U-Net (92%, 56%).

The malaria parasite Plasmodium falciparum invades, replicates within and destroys red blood cells in an asexual blood stage life cycle that is responsible for clinical disease and crucial for parasite propagation. Invasive malaria merozoites possess a characteristic apical complex of secretory organelles that are discharged in a tightly controlled and highly regulated order during merozoite egress and host cell invasion. The most prominent of these organelles, the rhoptries, are twinned, club-shaped structures with a body or bulb region that tapers to a narrow neck as it meets the apical prominence of the merozoite. Different protein populations localise to the rhoptry bulb and neck, but the function of many of these proteins and how they are spatially segregated within the rhoptries is unknown. Using conditional disruption of the gene encoding the only known glycolipid-anchored malarial rhoptry bulb protein, rhoptry-associated membrane antigen (RAMA), we demonstrate that RAMA is indispensable for blood stage parasite survival. Contrary to previous suggestions, RAMA is not required for trafficking of all rhoptry bulb proteins. Instead, RAMA-null parasites display selective mislocalisation of a subset of rhoptry bulb and neck proteins (RONs) and produce dysmorphic rhoptries that lack a distinct neck region. The mutant parasites undergo normal intracellular development and egress but display a fatal defect in invasion and do not induce echinocytosis in target red blood cells. Our results indicate that distinct pathways regulate biogenesis of the two main rhoptry sub-compartments in the malaria parasite.

Red blood cell (RBC) invasion by malaria merozoites involves formation of a parasitophorous vacuole into which the parasite moves. The vacuole membrane seals and pinches off behind the parasite through an unknown mechanism, enclosing the parasite within the RBC. During invasion, several parasite surface proteins are shed by a membrane-bound protease called SUB2. Here we show that genetic depletion of SUB2 abolishes shedding of a range of parasite proteins, identifying previously unrecognized SUB2 substrates. Interaction of SUB2-null merozoites with RBCs leads to either abortive invasion with rapid RBC lysis, or successful entry but developmental arrest. Selective failure to shed the most abundant SUB2 substrate, MSP1, reduces intracellular replication, whilst conditional ablation of the substrate AMA1 produces host RBC lysis. We conclude that SUB2 activity is critical for host RBC membrane sealing following parasite internalisation and for correct functioning of merozoite surface proteins.

Clustering analysis is a key technique for quantitatively characterizing structures in localization microscopy images. To build up accurate information about biological structures, it is critical that the quantification is both accurate (close to the ground truth) and precise (has small scatter and is reproducible).

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.

For its replication within red blood cells, the malaria parasite depends on a highly active and regulated lipid metabolism. Enzymes involved in lipid metabolic processes such as phospholipases are, therefore, potential drug targets. Here, using reverse genetics approaches, we show that only 1 out of the 19 putative phospholipases expressed in asexual blood stages of Plasmodium falciparum is essential for proliferation in vitro, pointing toward a high level of redundancy among members of this enzyme family. Using conditional mislocalization and gene disruption techniques, we show that this essential phosphoinositide-specific phospholipase C (PI-PLC, PF3D7_1013500) has a previously unrecognized essential role during intracellular parasite maturation, long before its previously perceived role in parasite egress and invasion. Subsequent lipidomic analysis suggests that PI-PLC mediates cleavage of phosphatidylinositol bisphosphate (PIP2) in schizont-stage parasites, underlining its critical role in regulating phosphoinositide levels in the parasite. IMPORTANCE The clinical symptoms of malaria arise due to repeated rounds of replication of Plasmodium parasites within red blood cells (RBCs). Central to this is an intense period of membrane biogenesis. Generation of membranes not only requires de novo synthesis and acquisition but also the degradation of phospholipids, a function that is performed by phospholipases. In this study, we investigate the essentiality of the 19 putative phospholipase enzymes that the human malaria parasite Plasmodium falciparum expresses during its replication within RBCs. We not only show that a high level of functional redundancy exists among these enzymes but, at the same time, also identify an essential role for the phosphoinositide-specific phospholipase C in parasite development and cleavage of the phospholipid phosphatidylinositol bisphosphate.

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.